Vaccination of Mice with Bacteria Carrying a Cloned Herpesvirus Genome Reconstituted In Vivo

Abstract

Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.Live, attenuated bacteria have a potential to serve as vaccine vectors for the oral delivery of foreign proteins (29) or DNA (11,13,23). Oral vaccination procedures do not require educated personnel, and lyophilized bacterial preparations can be kept and distributed at room temperature, without the costly and difficult necessity of keeping the preparations cold. Moreover, oral vaccination can be performed simultaneously on large numbers of subjects, which should make it an efficient strategy for livestock vaccination.Live antiviral vaccines are generally considered more efficient than subunit or inactivated vaccines because they are more likely to induce a broad range of immune responses to the expressed gene products and provide a better protection. Furthermore, since these vaccines replicate in the recipient, the immunity they confer should be long lasting (39). Therefore, a bacterial vaccination vector delivering an attenuated, yet infectious virus may present the basis for efficient vaccines that are easy to store, distribute, and administer.Herpesviruses are important pathogens for humans and livestock. We have previously shown that the large genomes of herpesviruses (up to 230 kb) can be cloned as bacterial artificial chromosomes (BACs) into Escherichia coli (1, 2, 24). Others have demonstrated that BAC DNA encoding the genome of a herpesvirus can induce specific protective immunity (35,36). However, in these experiments, the BAC DNA was isolated from bac...

Virus Progeny of Murine Cytomegalovirus Bacterial Artificial Chromosome pSM3fr Show Reduced Growth in Salivary Glands due to a Fixed Mutation of MCK-2

“…This was also shown by other groups (10,12,15,21,32) but was never addressed, although it stood in marked contrast to the high salivary gland titers observed when BALB/c mice were infected with MCMV strains not cloned as BACs (17,22,28,39). To find out whether this phenotype of the pSM3fr-derived virus was due to genomic differences between the MCMV Smith strain and the pSM3fr genome, we sequenced the complete genomes of pSM3fr, two independent pSM3fr-derived mutants (pSM3fr-⌬m157-⌬M27 and pSM3fr-⌬loxP) and a virus stock derived from pSM3fr-⌬m157-⌬M27.…”

“…This was also observed when pSM3fr-derived viruses were characterized in vivo (10,12,15,21,32) and stood in marked contrast to high salivary gland titers obtained after infection with MCMV strains not cloned as BACs (17,22,28,39). We could restore the phenotype of high virus titers in salivary glands by repairing the pSM3fr mutation and thus provide evidence that pSM3fr-derived virus shows an attenuated growth phenotype in salivary glands due to a mutation in MCK-2.…”

Targeted Deletion of Regions Rich in Immune-Evasive Genes from the Cytomegalovirus Genome as a Novel Vaccine Strategy

“…After mice were infected, their weight loss was monitored daily. At 16 weeks p.i., titers of virus-specific antibodies in sera of immunized mice were determined by indirect enzyme-linked immunosorbent assay (ELISA), as described previously (18). In brief, plates were coated with lysates from MCMV-infected MEF as the source of antigen or with uninfected MEF as the background control.…”

In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial cells

“…3,9,11 In vivo, E. coli has been used as vector for DNA vaccines by intramuscular, intraperitoneal or subcutaneous administrations. 12,13 Here, we explored the ability of not genetically modified L. lactis to deliver plasmid in vivo to mice. We showed that oral administration of lactococci carrying a BLG eukaryotic expression plasmid induces a local production of BLG in the small intestine.…”